Immobilisation and stabilisation of virus

ABSTRACT

The present invention relates to a method for immobilization and optional stabilization of viruses whilst retaining the viral biological activity and the use of immobilized virus in therapy. In particular, the immobilized virus relates to immobilized bacteriophage and their use as an antibiotic or bacteriostatic agent and in the treatment of antibiotic-resistant infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.10/512,635, filed on May 31, 2005, which is a U.S. National Phase of PCTApplication No. PCT/GB2003/001797, filed on Apr. 28, 2003, and whichclaims priority to British Application No. GB0209680.8, filed Apr. 27,2002, all of which are incorporated herein by reference in its entirety.

The present invention relates to a method for immobilising andoptionally stabilising viruses including bacteriophage, preferably to asolid phase substrate, for use in therapy, in particular as anantibiotic (bactericide) or bacteriostatic agent, in the treatment ofantibiotic-resistant superficial infections and use in vaccinations.

Bacteria have proven adept at developing resistance to newanti-microbial agents and so-called “super-bugs” are a cause of risingcosts spent on means to combat super-bug-related infections andfatalities in hospitals throughout the world. For example, the use ofantibiotics, whether in an individual patient or in a hospital with itsspecial environment and catalogue of micro-organisms, will destroyantibiotic-susceptible bacteria but permit the proliferation of bacteriathat are intrinsically resistant or that have acquired extra chromosomalresistance. Thus, the more antibiotics are used, the more resistantbacteria become.

Bacteria can survive on common hospital materials including cottonand/or polyester lab coats, privacy curtains and polyethylene splashaprons for anything up to seven weeks increasing the chance of spreadinginfection.

Indeed, common disinfectants used to sterilise hospital rooms andequipment are not sufficient to curb the spread of “super-bugs”.

Furthermore, no fundamentally new antibiotic has been discovered for atleast 30 years and there is no guarantee that new classes of antibioticswill be developed let alone even discovered in the next decade.

An alternative to antibiotics in the fight against “super-bugs” is theuse of bacteriophage. A bacteriophage is a water-borne virus thatinfects specific bacteria.

Virus particles vary in shape and size, from 0.02 to 0.3 um and containRNA or DNA, either double or single stranded, which forms the viralgenome. The viruses have a varied structure but the nucleic acid isalways located within the virus particle surrounded by a protein coat(capsid or shell). The complex of nucleic acid and protein (thenucleocapsid) may be the whole structure of the virus (for example 6, anRNA bacterial virus, or X174, a DNA bacterial virus) but structures thatare more complicated occur. The enveloped viruses may have lipid andprotein membranes around the capsid while the complex viruses possessnot only icosahedral heads but also helical tails with up to 20 proteinswithin the tail.

Bacteriophage on infection of their hosts can multiply by either a lyticor lysogenic pathway. Bacteriophage that can integrate their DNA intobacterial chromosomes are known as lysogenic bacteriophage, with theintegrated viral DNA replicating along with the host chromosome toproduce new integrated viral DNA copies. Alternatively, the virus mayreplicate freely to produce several hundred progeny particles. Lysis ofthe cells then releases this large number of free viruses, which arethen able to infect neighboring bacteria. Although, bacteriophage werefirst identified in 1917, studies in the West into their application inmedicine have been few and far between but studies have persisted andproved successful in Russia.

Even so, various problems with bacteriophage therapy remain. Forexample, although bacteriophage are easy to grow they are particularlyunstable and thus difficult to store. Bennett et al (1997) describe theimmobilisation of a Salmonella-specific bacteriophage by adsorption.They detail the passive adsorption of bacteriophage onto polystyrenesolid phases. However, this process is inefficient due to complexbacteriophages being immobilised via both “head” and “tail” groups. Thetail group is required to be free in order to recognise and infectspecific bacteria. Further, the adsorption process is reversible so thatadsorbed bacteriophage will desorb and release free bacteriophage. Theproduct of this process is described for use as a separation system forthe removal of specific bacteria from foods only and does not requirethe bacteriophage to be viable.

It is an object of the present invention to obviate and/or mitigate atleast some of the above disadvantages.

Broadly speaking, the present invention describes for the first time amethod for the immobilisation and optional stabilisation of viruseswhilst retaining the viral biological activity. Furthermore, itdocuments its use in therapy, for example the manufacture of medicaldevices comprising immobilised virus, such as bacteriophage, with theability to destroy specific resistant bacteria when, and only when, theyare present.

In a first aspect the present invention provides a device comprisingvirus immobilised to a substrate for medical application.

In a further aspect of the present invention there is provided a devicecomprising virus immobilised to a substrate for use as an antibiotic(bactericide) or bacteriostatic agent. Preferably, said virus is abacteriophage.

Immobilisation is understood to relate to a specific physicalimmobilization, such as by chemical bonding and is thereforedistinguished from any passive adherence of a virus to a substrate.

The term “virus” according to the present invention includesdouble-stranded or single-stranded RNA or DNA viruses, which infectcells of bacteria, plants and/or animals. These include viruses from thefollowing families of viruses: Iridoviridae, African swine fever virus,Poxyiridae, Parvoviridae, Reoviridae, Birnaviridae, Picornaviridae,Togaviridae, Flaviviridae, Rhabdoviridae, Bunyaviridae, Herpesviridae,Adenoviridae, Papovaviridae, Hepadnaviridae, Coronaviridae, Calicivirus,Arenaviridae, Paramyxoviridae, Orthomyxoviridae, Filoviridae,Retroviridae, Baculoviridae, Polydnaviridae, Nudaurelia (3 virus group,Nodaviridae, Caulimovirus, Geminivirus, Tomato spotted wilt virus group,Luteovirus, Machlovirus, Necrovirus, Sobemovirus, Tombusvirus,Tymovirus, Bromovirus, Cucumovirus, Ilarvirus, Alfafa mosaic virusgroup, Comovirus, Dianthovirus, Nepovirus, Pea enation mosaic virusgroup, Tobamovirus, Tobravirus, Hordeivirus, Potexvirus, Potyvirus,Carlavirus, Closterovirus, Totiviridae, Partitiviridae, Myoviridae,Styloviridae, Podoviridae, Tectiviridae, Plasmaviridae, Corticoviridae,Microviridae, Inoviridae, Cystoviridae and Leviviridae.

It should be understood that a virus may include viruses or infectiousagents, which do not fall into the above mentioned families, e.g., plantsatellite viruses, prions, baculoviruses and bacteriophage respectively.

The term “bacteriophage” according to the present invention isindicative of bacteriophage, which infect specific strains of bacteriae.g. salmonella, Escherichia coli, staphylococcus or pseudomonusbacteriophage.

The term “medical” according to the present invention is understood tomean the treatment or prevention of viral, bacterial or prion infectionsand/or contamination in humans, animals or plants. For example, in thecase of bacterial infections and/or contamination, treatment orprevention may be achieved by bacteriophage immobilised on a substrate.It will be understood to the skilled man that bacteriophage canrecognise and infect specific strains of bacteria. Thus, bacteriophageimmobilised to a substrate according to the present invention, may beutilised to fight strain-specific bacterial infections as a“bactericide” by inducing selective killing of bacteria through celllysis or as a “bacteriostatic agent” by inhibiting bacterial growth.Bacteriophage immobilised to a substrate may also be used as anantibacterial agent/disinfectant in order to “sterilise”bacterially-contaminated material.

The term “substrate” according to the present invention is understood tomean any solid phase material to which a virus may be immobilised. Forexample, said substrate may be a material which may be advantageouslyactivated to allow head-group specific binding of a virus, such ascomplex bacteriophage. Said substrate may take many forms, for example,nylon and any other polymer with amino or carboxyl surface groups,cellulose or other hydroxyl-containing polymer, polystyrene or othersimilar polymer, various plastics or microbeads including magneticparticles, biological substances. More preferably, said substrate ismade of a material commonly used in therapy/medicine. For example, nylonthread for use in surgery; plastics, lint or gauze material used todress open wounds; microbeads, which can be ingested; adhesives such ascyanoacrylates; and/or biological substances such as collagen orhyaluronic acid.

Immobilisation of virus to the substrate may be achieved in a number ofways. Preferably, viruses, such as bacteriophage, are immobilised viabonds, typically covalent bonds formed between the bacteriophage coatprotein and the substrate.

More preferably, bacteriophage are immobilised to the substrate viatheir head groups or nucleocapsid by activating the substrate before theaddition and coupling of bacteriophage.

The term “activated/activating/activation” according to the presentinvention is understood to mean the activation of a substrate byreacting said substrate with various chemical groups (leaving a surfacechemistry able to bind viruses, such as bacteriophage head or capsidgroups).

Activation of said substrate may be achieved by, for example,preliminary hydrolysis with an acid, preferably HCl followed by a washstep of water and an alkali to remove the acid. Preferably, said alkaliis sodium bicarbonate. Binding of viruses, for example bacteriophage,via their head groups is important. In the case of complex bacteriophagefor example, binding via head groups leaves the tail groups, which arenecessary for bacteria-specific recognition, free to infect, i.e., bindand penetrate a host bacterial cell. It will be understood that thismechanism of infection of a host cell is similar for many other virusesother than viruses that infect and multiply only in bacteria. Aplurality of viruses, e.g., various strain-specific bacteriophage, maybe immobilised to a substrate at any one time.

Coupling of viruses to a substrate is as a result of the formation ofcovalent bonds between the viral coat protein and the substrate such asthrough an amino group on a peptide, for example a peptide bond.“Coupling Agents” that aid this process vary, dependent on the substrateused. For example, for coupling to the substrate nylon or other polymerwith amino or carboxy surface groups the coupling agents carbodiimide orglutaraldehyde may be used.

For coupling to the substrate cellulose or other hydroxyl-containingpolymer the coupling agents vinylsulfonylethylene ether or triazine maybe used.

Coupling agents for the coupling of virus to the substrate polythene orother similar polymer include corona discharge or permanganateoxidation. Generally speaking, coupling agents for the coupling ofbacteriophage to a substrate include: S-Acetylmercaptosuccinicanhydride; S-Acetylthioglycolic acid N-hydroxysuccinimide ester; Adipicacid dihydrazide; 4-Azidobenzoic acid N-hydroxysuccinimide ester;N-(5-Azido-2-nitrobenzyloxy) succinimide; 6-(4˜Azido-2-nitrophenylamino)hexanoic acid N-hydroxysuccinimide ester; p-Azidophenacyl bromide;4-Azidosalicylic acid N-hydroxysuccinimide ester; Bromoacetic acidN-hydroxysuccinimide ester; 1,4-Butanediol diglycidyl ether;2-Diazo-3,3,3-trifluoroproprionic acid p-nitrophenyl ester; Diethylmalonimidate; 4,4′Diisothiocyanatostilbene-2,2′-disulfonic acid;Dimethyl adipimidate; Dimethyl 3,3′-dithiobispropionimidate; Dimethylpimelimidate; Dimethyl suberimidate; 4,4′-Dithiobisphenyl azide;Dithiobis(propionic acid N-hydroxysuccinimide ester); EthyleneGlycolbis-(succinic acid N-hydroxysuccinimide ester);4-Fluoro-3-nitrophenyl azide; p-Formylbenzoic acid N-hydroxysuccinimideester; Glutaraldehyde; 2-Iminothiolane; 6-(Iodoacetamide) caproic acidN-hydroxysuccinimide ester; Iodoacetic acid N-hydroxysuccinimide ester;3-Maleimidoacetic acid N-hydroxysuccinimide ester; 3-Maleimidobenzoicacid N-hydroxysuccinimide ester; 4-(N. Maleimido) benzophenone;compounds that protect proteins against dehydration, prolonged storageand other stresses. An example of such a compound is trehalose.

Trehalose and other similar agents including functional analogues areknown as stabilizing agents for a number of chemicals, living tissuesand even organisms, including viruses (Colaco et al., 1992; Crowe andCrowe 2000). Trehalose, a disaccharide, has been documented to beinvolved in the stabilisation of membranes and proteins in dry animalsand other anhydrobiotic organisms. Such organisms include, for example,dry baker's yeast Sacchoromyces cerevisiae, resurrection plants, cystsof certain crustaceans (including the brine shrimp Artemia) and manybacteria (Crowe and Crowe, 2000, Nature Biotech., 18, pp 145-146).Trehalose has also been shown to preserve mammalian cells duringfreezing (Beattie et al., 1997, Diabetes, 46, pp 519-523) and proteins(Colaco et al. 1992, Biotechnology, 10, pp 1007-1011) during drying.Trehalose has also been documented to be involved in the stabilisationof viruses in their native state by Bieganski et al., 1998, Biotechnol.Prog., 14, 615-620.

“Stabilization of active recombinant retroviruses in an amorphos drystate with trehalose” The present invention not only shows thattrehalose may be used to stabilise viruses in their native state but forthe first time shows that further stability results from trehalosetreatment of viruses immobilised by covalent attachment to a substrate.Thus, in a further aspect of the present invention there is provided useof trehalose for the further stabilisation of a device comprising virusimmobilised to a substrate according to the present invention and ashereinbefore described. Covalent immobilization (forming of a chemicalbond) of proteins as known, results in a substantial increase instability. The present inventors have also shown that this is true forbacteriophage by describing herein the stability of an insolublenylon/bacteriophage co-polymer of considerable molecular weight.

Thus, according to the present invention said virus (es) immobilised tosaid substrate may be for example, coated with trehalose by, forexample, dipping into a solution of trehalose before drying and storagesuch that the virus (es) maintain their viability and infectivity.

Further applications of the present invention may include the treatmentof MRSA; food poisoning, wherein bacteriophage may be immobilised on asubstrate such as microbead suspension, which can be ingested; or in thedecontamination of hospital equipment/surfaces; Prevention of infectionbymethicillin-resistant Staphylococcus aureus or VISA (vancomycininsensitive S. aureus) by immobilization of appropriate bacteriophageonto, for example, sutures or wound dressings; prevention of specificpathogen entry through catheters and similar devices by immobilizationof appropriate bacteriophage onto the surface of the device; treatmentof pulmonary infection such as tuberculosis with, for example,micro-particles of about 10 microns diameter with appropriatebacteriophage immobilized onto them, by inhalation; treatment ofinfections such as meningitis, for example, by injection of microparticles with appropriate bacteriophage strains immobilized; treatmentof gastrointestinal infections, for example, by particles or gelscontaining appropriate immobilized bacteriophage; treatment of bacterialplant diseases; elimination of, for example, E. coli in cattle byincorporation into the diet of immobilized bacteriophage; incorporationof appropriate bacteriophage into food wrapping materials to prevent oreliminate contamination by organisms causing food poisoning;incorporation of appropriate bacteriophage into paints to preventcontamination of surfaces in, for example, hospitals, or farms;treatment of surfaces of air-conditioning units with appropriateimmobilized bacteriophage to prevent, for example, Legionellacontamination.

The present invention may also be used for the purposes of vaccination.For example, the present invention may be used to immobilize any live,infectious virus, which could be then used to vaccinate populations atrisk. This may be particularly useful where no vaccine exists for aparticular virus and any form of prevention of viral infection would beuseful. Thus, a virus as hereinbefore described may be immobilised andused directly to vaccinate reducing the time taken to develop a morestandard attenuated virus. It will be understood that such vaccinationsmay only be conducted, especially with regard to humans, in extremecases. Without wishing to be bound by theory, the action of theimmobilised virus used to vaccinate would be such that the immobilizedvirus would be unable to reach its target cells from the vaccinationsite; preventing the immobilised virus infecting the patient.

It will be understood, therefore, that immobilised HIV virus would notbe able to be used for vaccination in this regard. Preferably, theimmobilised virus is kept at the inoculation site for longer thanconventional vaccines allowing a better immune response to be raised.

Advantageously, the present inventor has shown that the presentinvention of immobilising viruses substantially reduces or eliminatesfree, unimmobilised viruses.

In a further aspect, the present invention provides a method ofpreparing a device comprising a substrate having virus immobilisedthereon, said method comprising the steps of: a) activating thesubstrate so as to enable virus to bind thereto; b) mixing the modifiedsubstrate with virus and a coupling agent to aid the binding of virus tothe substrate.

In a yet further aspect said method comprises the further step of: c)mixing the device with a stabilising agent that maintains the viabilityand infectivity of the virus bound to the modified substrate when saidmodified substrate is exposed to dehydration, prolonged storage and/orother stresses.

It should be understood that the term “activating” is as hereinbeforedefined.

Preferably, the stabilising agent according to the present invention istrehalose or other agent such as known heat shock proteins known in theart that protect proteins or viruses against dehydration, prolongedstorage and other stresses.

In a preferred embodiment of the present invention, said device mixedwith a stabilising agent may be dried, allowing prolonged storage ofsaid device whilst maintaining the infectivity and viability of thevirus.

The present invention will now be further described by way of example,with reference to the following methods and figures in which:

FIG. 1—Graphical Representation of Wound Model 1

The graph depicts the activity over three days of nylon strips with orwithout bacteriophage in killing Staphylococcus aureus bacterium presenton the surface of raw pork to simulate a surgical wound. The activityrepresents the average of three replicates, score + or −, wherein +depicts the clearing of bacterium and − depicts the bacterium remainingcloudy.

FIG. 2—Graphical Representation of Wound Model 2

Activity is measure over 9 days and is scored as in FIG. 1. Nylon+/−bacteriophage is inserted into a wound in fresh raw pork, which isreplaced with fresh tissue every three days.

FIG. 3—Graphical Representation of Resistance of ImmobilisedBacteriophage to proteolytic activity

Series 1-control;

Series 2-0.1 g/l trypsin;

Series 3-0.5 g/l trypsin;

Series 4-2.5 g/l trypsin

FIG. 4

Graphical representation of numbers of bacteriophage immobilised onactivated nylon. The reduction of phage numbers with time is depicted.

FIG. 5

As for FIG. 4, repeated with larger strips of activated nylon (5×1 cm)

FIG. 6

Schematic drawing of Wound Models 1 and 2 (depicted in FIGS. 1 and 2respectively).

FIG. 7—Graphical Representation of Infection of Animal Cells byAdenovirus

The graph depicts the infectivity of free adenovirus, and adenovirusimmobilised onto nylon on HEK 293 cells.

METHODS

Propagation: An overnight subculture of bacteria was adjusted to a cellconcentration of 1.5×109 cells/ml. 0.1 ml of this was mixed with 1×105pfu (plaque forming units) of bacteriophage. After incubation at 37 Cfor 20 minutes the mixture was poured onto 1.5% LB agar, 0.7% LB agarwas layered over this and allowed to set. Plates were incubated at 37 Cfor 12 hours. Almost confluent bacteriophage plaques were formed.Bacteriophage were harvested by adding 5 ml of sterile bacteriophagesuspension buffer and shaking. The bacteriophage suspended in the bufferwere purified by centrifugation and filtration through a 100 kDa cut-offfilter to remove bacterial protein. Yield was 1×109 pfu/ml.

Plaque assays: for the presence of bacteriophage were carried out by thetwo layer plate assay as described for propagation.

Immobilisation: Nylon strip 8×1 cm was used.

Activation: Nylon was activated by preliminary hydrolysis with 4M HClfor 2.5 minutes at 70° C., washed in distilled water and 0.1M sodiumbicarbonate to remove acid.

Coupling to Nylon or Other Polymer with Amino or Carboxyl Surface Groups

i) Carbodiimide as a Coupling Agent.

After this brief acid hydrolysis of the nylon surface the sample iswashed with dimethylformamide (DMF) and 20 mM1-cyclohexyl-3-[2-morpholinoethyl]-carbodiimidemethyl-p-toluenesulphonate is added. The solution is stirred for 90 minutes, and thenthe nylon is washed with DMF. The activated nylon is stirred overnightwith bacterophage in suitable buffer, and then washed to remove unboundbacteriophage.

ii) Glutaraldehyde as a Coupling Agent

After a brief acid hydrolysis of the nylon surface the sample is washedwith 0.1M bicarbonate buffer pH9.4 and incubated with 10% glutaraldehydein 0.1M bicarbonate buffer. The surface was then washed in bicarbonatebuffer and distilled water before being incubated overnight withbacteriophage in a suitable buffer.

Coupling to Cellulose or Other Hydroxyl-Containing a Polymer. i)Vinylsulfonylethylene Ether

Vinylsulfonyl groups can be introduced into hydroxyl-containing polymersby treatment of the polymer with vinyl sulfone at pH11. The activatedpolymer is stirred overnight with bacteriophage in suitable buffer, andthen washed to remove unbound bacteriophage.

ii) Triazine Addition

Cellulose or a modified cellulose (about 10 g) is added to 50 ml ofacetone/water (1:1) containing 1 g 2-amino-4,6-dichloro-s-triazine at 50and stirred for 5 minutes. Then 20 ml of 15% (w/v) aqueous sodiumcarbonate to which 0.6 vol. of 1M HCl has been added is poured into thereaction mixture. Concentrated HCl is then added to bring the mixture pHbelow 7. The amino-chloro-s-triazine substituted cellulose is washedwith acetone/water, then water and finally with 0.05M phosphate bufferas pH7.0.

The coupling reaction with the bacteriophage is carried out at pH8.0 in0.05M phosphate buffer by stirring for 12 to 18 hours.

Coupling to Polythene or Other Similar Polymer 1. Corona Discharge

Polythene was exposed to a corona discharge for about 1 second;bacteriophage dehydrated in the presence of trehalose was dusted ontothe treated surface immediately.

2. Permanganate Oxidation.

Polythene was exposed to concentrated potassium permanganate solution,for several hours, washed with distilled water and immediately treatedwith bacteriophage in trehalose or other stabilizing agent.

Example 1

1. Bacteriophage PI with Escherichia coli 11291 The nylon/bacteriophagepreparation was challenged with 50 ml of bacterial culture at about1×108 cells/ml. After incubation the culture was assayed by the twolayer plaque assay.

Example 2 Bacteriophage a Against E. Coli

In this experiment the number of pfu's used in the preparation of theimmobilised system is compared with the number of pfu's observed whenthe immobilised system is challenged with the bacteria.

Plaques Free bacteriophage 8 40 35 No bacteriophage 0 0 0 Immobilisedbacteriophage 7 23 17

The numbers of bacteriophage plaques arising from the immobilisedsystems with this bacteriophage-bacteria combination shows:

-   -   that immobilised bacteriophage are viable and infective.    -   a relationship between free bacteriophage numbers used in the        preparation and the number of bacteriophage produced by        immobilised systems.

Example 3 Unknown Bacteriophage Against Staphylococcus aureus

Bacteriophage was isolated by incubating a lawn of S. aureus withcontaminated water which had been filtered through a 0.18 filter toremove bacteria. Where a plaque formed indicated the presence of a lyticbacteriophage. This was isolated and grown as previously described.

Dilution Plaques expected Plate Count 1 1 0 2 10 0 3 100 10 4 1000 100 510000 1000 6 100000 10000

Example 4 Effect of Trehalose on Viability

The experimental system was as previously described except that thenylon/bacteriophage preparations were dipped into a trehalose solutionof various concentrations and dried before assay (Nylon/bacteriophagepreparations previously described were stored in buffer for 24 to 48hours before use) dried preparations were used 72 hours later.

Plaques Free bacteriophage 24 No bacteriophage 0 Immobilisedbacteriophage 21 Trehalose 1% 27 Trehalose 0.5% 18 Trehalose 0.1% 24Trehalose 0.05% 21

The data indicate that trehalose enables immobilised bacteriophage towithstand dessication and storage for at least 72 hours withoutsignificant loss of viability and infectivity. The free bacteriophage,no bacteriophage and immobilised bacteriophage samples were controls nottreated to dessication and storage.

Dilution Plaques expected Plate count 1 1 1 2 10 1 3 100 15 4 1000 250 510000 500 6 100000 1000

The data show that significant numbers of viable and infectivebacteriophage have been immobilised on the nylon sheet. There is also adose/response relationship between the estimated numbers ofbacteriophage immobilised and the plaques formed from the immobilisedsystem.

Validation of Washings:

The washings from the nylon/bacteriophage reaction were assayed by thetwo layer plaque assay to determine the rate and efficiency of removalof free bacteriophage (those that did not form covalent attachment as aresult of the chemistry).

Washing pfu/ml 1 10000 2 100 3 8 4 0 5 0 6 0

Example 5 Bacteriophage NCIMB 9563 (ATCC6538-B) against Staphylococcusaureus in the Presence of Tissue

Bacteriophage 9563 was grown as previously described, immobilized ontonylon membrane, washed to remove unbound bacteriophage and testedagainst a strain of S. aureus in two experimental situations.

1. Does the Presence of Animal Tissue Affect the Response of theImmobilised Bacteriophage?

The nylon membrane with immobilized bacteriophage was placed in a flaskwith 50 ml of S. aureus growth medium and 10 g of macerated beef, tomodel a wound situation, the flask was inoculated with 2×108 bacterialcells and incubated for 24 hours at 37 C. Samples were tested for thepresence of bacteriophage by the two layered assay previously described.The results showed that the macerated tissue did not significantlyaffect the infection of S. aureus by immobilized bacteriophage 9536.

Dilution Plaques expected Plate count 1 1 0 2 10 0 3 100 10 4 1000 100 510000 1000 6 100000 10000

The “plaques expected” is based on the estimated number of bacteriophageimmobilized and their expected subsequent propagation in the bacterialculture. The result implies that under the conditions of the experimentabout 10% of the immobilized bacteriophage infected bacteria or, morelikely, that 10% of the inoculated bacteria came into contact with thenylon membrane.

2. Does the Immobilized Bacteriophage have an Effect in a Wound Model?

A series of two-centimeter cuts were made in a beef or pork slice andeach was inoculated with 2×108 bacterial cells. Sections of nylonmembrane with immobilized bacteriophage 9536 were inserted into ten ofthe cuts, nylon membrane without bacteriophage into another ten, and tenleft untreated.

After 24 hours visible growth was evident in cuts left untreated ortreated with nylon only, but no growth was seen in cuts treated withimmobilized bacteriophage. This indicates that immobilized bacteriophageis effective in preventing bacterial growth in the presence of muscletissue.

Example 6 1. Wound Models

In a hypothetical clinical situation where sutures with immobilisedbacteriophage had been used, the longer the phage remained active thegreater the protection given. Although the major period of contaminationwould be during and immediately after surgery, the material would not beactivated until contact between target bacteria and the suture occurred.

Initial experiments used surface contact with fresh, raw pork tosimulate a surgical wound—See FIG. 6 a.

The assay involved incubating the exposed nylon/phage strips with thetargets. aureus (8588) cultures. Exposed strips were sterilized withchloroform (2.5%) to prevent contamination from other bacteria presenton the pork surface. The target bacterium was either cleared (+result)or remained cloudy (−result).

Conclusions:

Activity is substantially retained for three days in contact with“wound”—see FIG. 1.

Wound Model 2

In this test, the strips (nylon/phage) were inserted into a wound infresh raw pork, which was replaced with fresh tissue every threedays—see FIG. 6 b.

Results are Depicted in FIG. 2 Conclusions:

The activity was retained at a high level for 6 to 7 days. After thatperiod, one or more of the replicate strips failed to clear the S.aureus culture. This result suggests that a single presentation wouldprovide protection for longer than the likely time sutures would beused, and longer than the time in which wound dressings would be leftunchanged.

Trypsin Digest

The aim of this was to determine how resistant phage were to proteolyticdegradation, a possible stress in certain situations. Two experimentalapproaches were used, free bacteriophage were tested to determine theirsusceptibility to tryptic inactivation and immobilised bacteriophagewere tested, to determine any protective effect from the immobilization.

Free phage test 1.

Three concentrations of trypsin were used:

0.1 g/i0.5 g/l2.5 g/l

Tests were carried out in universal tubes with 5 ml of stock phage.

Samples were diluted and assayed for viable phage by standard plaqueassay.

Results are Depicted in FIG. 3 Conclusions:

The trypsin at these concentrations and times was without effect on thefree phage.

This experiment was repeated three times with similar outcomes.

The immobilised phage is at least as resistant as the free phage toproteolytic inactivation.

This result has implications for possible oral administration ofbacteriophage.

Numbers of Phage Immobilised

Two approaches have been adopted, one is to determine the residual phageafter immobilization and the other electron microscopy.

1. Activated nylon was added to phage solution of known concentrationand the reduction of phage numbers with time measured by plaque assay.

Results are Depicted in FIG. 5

The area of the strips was 5×1 cm, with the number of phage immobilisedbeing 7×107 per strip. This gives a density of one phage per 15, u2,electron microscopy methods give a similar density (7 to 15 u spacing).The size of phage is about 0.3 u, so the surface density could beincreased.

Example 7 Production of Lytic Bacteriophage Active Against PathologicalStrains of S. aureus Method

A lytic bacteriophage isolated from environmental sources was treatedwith mutagenic chemicals to alter the binding capability of thebacteriophage towards its target bacterium. Random mutagenesis can becarried out by any standard method, in the case reported below this wastreatment with hydroxylamine (4%).

Result

Plaques Methicillion PFGE with Antibiogram MIC Phage type profile mutantPuSuTe (MtCxErCp) R 932/77ih/ PF108a None 83A/84IH/ 85/90+ PnMtCx R 75wPF15a Confluent PnMtCxErCp (Im) R NT PF15b Confluent PnMtCxErClCpKmTb R83Cw/29ih/ PF16a Confluent 75w/77w/ 83Aw PnMtCxImErClCpKm R 83Cih/29ih/PF16m Confluent 52ih/75w/ 77/83Aw

Conclusions PF15a and b correspond to EMRSA type 15, which accounts forabout 70% of hospital-acquired infections. PF16a and M correspond toEMRSA type 16 which accounts for about 20% of such infections. Thisphage would be effective against 90% of all hospital-acquired infectionsin the UK.

Example 8 Activity of Immobilised Adenovirus Background

Method Nylon samples were activated with carbodiimide as previouslyindicated and reacted with Adeno-X-LacZ viral stock (BD Biosciences PaloAlta, USA). The samples were washed 5 times, then introduced into wellsof a 12 well assay plate. Healthy HEK 293 cells (5×105 cells/ml) wereseeded into each well, together with positive and negative controls. Thepositive control consisted of un-immobilised

Adeno-XLacZ virus (“free virus”). Cells were cultured in DMEM+10% FetalBovine serum medium. Several dilutions of the stock virus were used(10-2 to 10-6). Plates were incubated at 37 C in 5% CO2/air for 48hours. The medium was removed cells air dried for 5 minutes, then fixedby adding 1 ml ice-cold methanol to each well. After 10 minutes themethanol was removed and wells rinsed three times with 1 ml Phosphatebuffered saline (PBS)+1% bovine serum albumin (BSA).

Anti-hexon antibody (BD Biosciences Palo Alta, USA) was diluted 1:1000,and 0.5 ml added to each well, incubated for 1 hour at 37 C withshaking.

The antibody was removed, wells rinsed three times with PBS+1% BSA. RatAnti-Mouse antibody (Horse radish peroxidase conjugate), diluted 1:500,was added to each well (0.5 ml), incubated with shanking for a further 1hour at 37 C, then rinsed three times with PBS=1% BSA (1 ml). DABworking solution was prepared by dilution of the 10× concentrate withStable peroxidase buffer (BD Biosciences Palo Alta, USA).

0.5 ml of DAB working solution was added to each well and incubated atroom temperature for 10 minutes. The DAB solution was removed and 1 mlPBS added.

Immunisation Protocol

The Adeno-X-LacZ viruses were immobilised onto nylon spheres ofapproximately 10 microns diameter, using the carbodiimide protocolpreviously described and washed five times. About 0.25 ml of asuspension of the nylon spheres with immobilised adenovirus in completeFreunds Adjuvant were injected into Balb C mice, and a further 0.3 mlinjected after three weeks.

Mice were bled 14 days after the final injection and the serum testedfor the presence of antibody to the adenovirus.

Adeno-X-LacZTM immobilised onto nylon was treated with diluted serumfrom the mice instead of the Anti-hexon antibody, otherwise the protocolwas as described in the previous section.

Sampling Wells with cell layers were viewed using an inverted microscopeunder 10, 20 and 40× objectives with bright field and phase contrast.

Nylon squares were mounted on slides and viewed with bright field andphase contrast microscopy under 10, 20 and 40× objectives.

Darkly brown-stained cells were interpreted as infected (+). Unstainedcells as infected Results The immobilised adenovirus was incapable ofinfecting the HEK 293 cells, although the free virus was infective. Thisindicates that immobilization prevents phagocytosis of virus particlesby animal cells, and hence infection by the virus.

The immobilised adenovirus, when injected into rats, gave an immuneresponse indicating that the adenovirus protein was present on thesurface of the nylon.

1. A method for treating or preventing viral, bacterial, or prioninfection and/or contamination in a subject in need thereof, comprisingadministering to said subject a device comprising bacteriophageimmobilised to a substrate via covalent bonds formed between thebacteriophage head group or nucleocapsid and the substrate, such thatthe bacteriophage retains infectivity.
 2. The method of claim 1, whereinsaid infection is a viral infection.
 3. The method of claim 1, whereinsaid infection is a bacterial infection.
 4. The method of claim 1,wherein said infection is a prion infection.
 5. The method of claim 1,wherein immobilisation confers increased stability to saidbacteriophage.
 6. The method of claim 1, wherein said device has aplurality of different strain-specific bacteriophage immobilisedthereon.
 7. The method of claim 1, wherein said substrate is a materialwhich is activated to allow head-group specific binding of abacteriophage.
 8. The method of claim 1, wherein said bacteriophage isimmobilised via its head group leaving the tail group free.
 9. Themethod of claim 1, wherein immobilisation of bacteriophage to asubstrate via a covalent bond is aided by the addition of a couplingagent.
 10. The method of claim 9, wherein said coupling agent iscarbodiimide or glutaraldehyde for coupling of bacteriophage to thesubstrate nylon or other polymer with amino or carboxy surface groups;vinylsulfonylethylene ether or triazine for coupling of bacteriophage tothe substrate cellulose or other hydroxyl-containing polymer;permanganate oxidation for the coupling of bacteriophage to thesubstrate polythene or other similar polymer.
 11. The method of claim 7,wherein said activation is carried out by corona discharge.
 12. Themethod of claim 1, wherein said immobilised bacteriophage is treatedwith a compound that protects proteins against dehydration, prolongedstorage and other stresses and wherein said immobilised bacteriophagedisplays increased viability and an infectivity when treated incomparison to untreated bacteriophage.
 13. The method of claim 12,wherein said compound is trehalose.
 14. The method of claim 1, whereinsaid device is a bandage, suture, compress or wound-dressing, implant,bead, or plaster.
 15. A method for treating or preventing viral,bacterial, or prion infection and/or contamination in a subject in needthereof, comprising administering to said subject a device comprisingbacteriophage immobilised to a substrate wherein the substrate has beenactivated using corona discharge and the bacteriophage is immobilised tothe substrate via covalent bonds formed between the bacteriophage headgroup or nucleocapsid such that the bacteriophage retains infectivity.16. The method of claim 15, wherein said infection is a viral infection.17. The method of claim 15, wherein said infection is a bacterialinfection.
 18. The method of claim 15, wherein said infection is a prioninfection.
 19. The method of claim 15, wherein immobilisation confersincreased stability to said bacteriophage.
 20. The method of claim 15,wherein said device has a plurality of different strain-specificbacteriophage immobilised thereon.
 21. The method of claim 15, whereinimmobilisation of bacteriophage to a substrate via a covalent bond isaided by the addition of a coupling agent.
 22. The method of claim 21,wherein said coupling agent is carbodiimide or glutaraldehyde forcoupling of bacteriophage to the substrate nylon or other polymer withamino or carboxy surface groups; vinylsulfonylethylene ether or triazinefor coupling of bacteriophage to the substrate cellulose or otherhydroxyl-containing polymer; permanganate oxidation for the coupling ofbacteriophage to the substrate polythene or other similar polymer. 23.The method of claim 15, wherein said immobilised bacteriophage istreated with a compound that protects proteins against dehydration,prolonged storage and other stresses and wherein said immobilisedbacteriophage displays increased viability and an infectivity whentreated in comparison to untreated bacteriophage.
 24. The method ofclaim 23, wherein said compound is trehalose.
 25. The method of claim15, wherein said device is a bandage, suture, compress orwound-dressing, implant, bead, or plaster.